CAJ | 학술논문

采用CTAB法、改进SDS-蛋白酶K法、SDS-饱和氯化钠法和提取试剂盒对喙尾琵甲的肌肉、虫卵和幼虫进行DNA提取,比较了DNA的提取和保存质量,并对AFLP试验的酶切时间、预扩增产物稀释倍数和选择性扩增引物量等关键因子进行试验和优化.结果表明:4种方法对肌肉组织提取的DNA质量都较好;3种传统方法提取的DNA的保存时间长于试剂盒提取的DNA;使用EcoR Ⅰ/Mse Ⅰ内切酶组合,10 U EcoR Ⅰ和2 U Mse Ⅰ分两步各酶切2 h,T4连接酶3 h连接后,以20倍稀释的预扩增产物和5 ng/35 ng的E+3/M+3引物量进行选择性扩增,建立了喙尾琵甲AFLP分子标记体系.
채용CTAB법、개진SDS-단백매K법、SDS-포화록화납법화제취시제합대훼미비갑적기육、충란화유충진행DNA제취,비교료DNA적제취화보존질량,병대AFLP시험적매절시간、예확증산물희석배수화선택성확증인물량등관건인자진행시험화우화.결과표명:4충방법대기육조직제취적DNA질량도교호;3충전통방법제취적DNA적보존시간장우시제합제취적DNA;사용EcoR Ⅰ/Mse Ⅰ내절매조합,10 U EcoR Ⅰ화2 U Mse Ⅰ분량보각매절2 h,T4련접매3 h련접후,이20배희석적예확증산물화5 ng/35 ng적E+3/M+3인물량진행선택성확증,건립료훼미비갑AFLP분자표기체계.
The study compared the quality of DNA extracting from muscles, eggs and larvae of Blaps rhynchopetera with CTAB method, improved SDS-protease-K method, SDS-NaCl method and DNA extraction Kit. Some key factors affecting the time of digestion, dilution of pre-amplification product and the amount of selective amplification primer were studied and an optimized AFLP reaction system of B. rhynchopetera was established. High quality genomic DNA could be isolated using four methods from this beetle muscles. DNA extracted with traditional extraction method could keep for longer time than DNA extraction Kit did. The optimal reaction time for enzyme digestion(EcoR Ⅰ/Mse Ⅰ) was 2 hours for each. Products of the pre-amplification diluted 20 fold and 5 ng/35 ng(E+3/M+3) was the best amount of the selective amplification primer.